Concerted use of restriction endonucleases and DNA ligases allows in vitro recombination of DNA sequences. The recombinant DNA generated by restriction and ligation may be amplified in an appropriate microorganism such as E. coli, and used for diverse purposes including gene therapy. However, the restriction-ligation approach has two practical limitations: first, DNA molecules can be precisely combined only if convenient restriction sites are available; second, because useful restriction sites often repeat in a long stretch of DNA, the size of DNA fragments that can be manipulated are limited, usually to less than about 25 kilobases.
Homologous recombination, generally defined as an exchange between homologous segments anywhere along a length of two DNA molecules, provides an alternative method for engineering DNA. In generating recombinant DNA with homologous recombination, a microorganism such as E. coli, or a eukaryotic cell such as a yeast or vertebrate cell, is transformed with exogenous DNA. The center of the exogenous DNA contains the desired transgene, whereas each flank contains a segment of homology with the cell's DNA. The exogenous DNA is introduced into the cell with standard techniques such as electroporation or calcium phosphate-mediated transfection, and recombines into the cell's DNA, for example with the assistance of recombination-promoting proteins in the cell.
In generating recombinant DNA by homologous recombination, it is often advantageous to work with short linear segments of DNA. For example, a mutation may be introduced into a linear segment of DNA using polymerase chain reaction (PCR) techniques. Under proper circumstances, the mutation may then be introduced into cellular DNA by homologous recombination. Such short linear DNA segments can transform yeast, but subsequent manipulation of recombinant DNA in yeast is laborious. It is generally easier to work in bacteria, but linear DNA fragments do not readily transform bacteria (due in part to degradation by bacterial exonucleases). Accordingly, recombinants are rare, require special poorly-growing strains (such as RecBCD-mutant strains) and generally require thousands of base pairs of homology. Thus, improved methods of promoting homologous recombination in bacteria are needed.
In eukaryotic cells, targeted homologous recombination provides a basis for targeting and altering essentially any desired sequence in a duplex DNA molecule, such as targeting a DNA sequence in a chromosome for replacement by another sequence. The approach may be useful for treating human genetic diseases.
Homologous recombination has been used to create knock-out mutants and transgenic animals, and thereby has played a critically important role in understanding gene function. Transgenic animals are organisms that contain stably integrated copies of genes or gene constructs derived from another species in the chromosome of the transgenic animal. These animals can be generated by introducing cloned DNA constructs of the foreign genes into totipotent cells by a variety of methods, including homologous recombination.
Currently, methods for producing transgenics have been performed on totipotent embryonic stem cells (ES) and with fertilized zygotes. ES cells have an advantage in that large numbers of cells can be manipulated in vitro before they are used to generate transgenics. Alternatively, DNA can also be introduced into fertilized oocytes by micro-injection into pronuclei, or injection into the germline of organisms including C. elegans or Drosophila species.
Several methods have been developed to detect and/or select for targeted site-specific recombinants between vector DNA and the target homologous chromosomal sequence (Capecchi, Science 244:1288, 1989). Cells that exhibit a specific phenotype after recombination, such as occurs with alteration of the hypoxanthine phosphoribosyl transferase (hprt) gene, can be obtained by direct selection on the appropriate growth medium. Alternatively, a selectable marker such as neomycin resistance can be incorporated into a vector under promoter control, and successful transfection can be scored by selecting G418-resistant cells (Joyner et al., Nature 338:153, 1989). Numerous other selection procedures have been described (Jasin and Berg, Genes and Development 2:1353, 1988; Doetschman et al., Proc. Natl. Acad. Sci. U.S.A. 85:8583, 1988; Dorini et al., Science 243:1357, 1989; Itzhaki and Porter, Nucl. Acids Res. 19:3835, 1991). Unfortunately, exogenous sequences transferred into eukaryotic cells undergo homologous recombination only at very low frequencies, even when very long homology regions are present (Koller et al., Proc. Natl. Acad. Sci. U.S.A., 88:10730, 1991, and Snouwaert et al., Science 257:1083, 1992). Thus, large numbers of cells must be transfected, selected, and screened in order to generate a correctly targeted homologous recombinant.